Molecular Ecology Resources (2013) doi: 10.1111/1755-0998.12082 Advancing nematode barcoding: A primer cocktail for the cytochrome c oxidase subunit I gene from vertebrate parasitic nematodes SEAN. W. J. PROSSER,* MARIA. G. VELARDE-AGUILAR,† VIRGINIA. LEON-R EGAGNON† and PAUL.D.N.HEBERT* *Biodiversity Institute of Ontario, University of Guelph, Guelph, Ontario N1G 2W1, Canada, †Estacion de Biologıa Chamela, Instituto de Biologıa, Universidad Nacional Autonoma de Mexico, San Patricio, Jalisco 48980, Mexico Abstract Although nematodes are one of the most diverse metazoan phyla, species identification through morphology is diffi- cult. Several genetic markers have been used for their identification, but most do not provide species-level resolution in all groups, and those that do lack primer sets effective across the phylum, precluding high-throughput processing. This study describes a cocktail of three novel primer pairs that overcome this limitation by recovering cytochrome c oxidase I (COI) barcodes from diverse nematode lineages parasitic on vertebrates, including members of three orders and eight families. Its effectiveness across a broad range of nematodes enables high-throughput processing. Keywords: barcoding, identification, nematodes, primers Received 19 September 2012; revision received 9 November 2012; accepted 13 November 2012 high phenotypic plasticity (Coomans 2002; Nadler 2002), Introduction the absence of clear diagnostic characters (Wijova et al. Roundworms (Nematoda) are known to be among the 2005; Derycke et al. 2008) or their restriction to adults in most physiologically and ecologically diverse of meta- the numerous groups in which larvae are more often zoan phyla, occupying habitats from the deep sea to encountered (Anderson 2000). Given these constraints, deserts, and from the tropics to polar permafrost (Brown there is recognition that molecular techniques are critical et al. 1949, 1950; De Ley 2006; Dailey 2009; Asbakk et al. for taxonomic progress (Godfray 2002; Blaxter 2003). 2010; Vanreusel et al. 2010). The phylum includes free- Indeed, there are now online databases, such as NemA- living, parasitic, mutualistic, opportunistic and symbiotic TOL (http://nematol.unh.edu/), that are dedicated to taxa (Ott et al. 1991; Clarke 2008) and provides a useful organizing and storing ecological and molecular data of model system for the study of human diseases (Fire et al. nematodes. 1998; Barr 2005; Jadiya et al. 2011) and a tool for ecosys- Several genetic markers have been used for nematode tem surveillance (Sambongi et al. 1999; Marcogliese 2005; identification, including small and large subunit ribo- Ekschmitt & Korthals 2006; Wu et al. 2010; Denver et al. somal DNA (SSU and LSU respectively), the internal 2011; Hoess et al. 2011; Palm et al. 2011). However, nema- transcribed spacer (ITS) region of ribosomal DNA and todes are also a scourge as many species cause disease in cytochrome c oxidase subunit I (COI) (Blaxter et al. 1998; crops, livestock and humans (Hodda & Cook 2009; Man- Floyd et al. 2002; Subbotin et al. 2008; Elsasser et al. 2009; guin et al. 2010). Despite their importance, the taxonomy Ferri et al. 2009; Siddal et al. 2012). The ribosomal DNA of nematodes is poorly studied. Species-level identifica- small subunit (SSU) was the first marker used, and tion has traditionally relied on detailed morphological successfully delineated some nematodes but failed to analysis, a task requiring considerable expertise (Coo- completely explain previous observations based on mor- mans 2000) given the morphological conservatism and phology (Blaxter et al. 1998). As the use of SSU was small size of nematodes (Creer et al. 2010; Powers et al. expanded, it was discovered that the SSU barcode failed 2011). Aside from being time-consuming, morphology- to separate many species of nematodes and was better based identifications are often problematic because of suited for order or family-level discrimination (De Ley et al. 2005). The ribosomal DNA large subunit (LSU) Correspondence: Sean W. J. Prosser, Fax: 519-824-5703; was the second marker used in an attempt to develop E-mail: [email protected] a nematode phylogenetic classification system, but © 2013 Blackwell Publishing Ltd Table 1 Nematode specimens used in this study. Taxa identified using morphology. Classification follows Hodda (2011) unless indicated by *, in those cases classification follows 2 Hodda (2007); ND: No Data S. W. J. PROSSER Number of specimens studied (successfully Order Family Genus sequenced) Host species Locality Collection date Panagrolaimida Rhabdiasidae Rhabdias sp. 1 1 (1) Smilisca Colima: Hwy Colima 7 July 2008 baudinii -Minatitlan ET AL. Panagrolaimida Rhabdiasidae Rhabdias sp. 2 4 (4) Rana sp. Nayarit: S of Hyw Barranca 25 June 2009 del Oro: Barranqueno~ bridge Panagrolaimida Rhabdiasidae Rhabdias sp. 3 5 (5) Rhinella marina Colima: Comala 7 July 2008 Panagrolaimida Rhabdiasidae Rhabdias lamothei 4 (4) Leptodeira sp. Colima: Hyw 98 Minatitlan 8 July 2008 -Manzanillo Rhabditida Molienidae* Oswaldocruzia sp. 9 (9) Phrynohyas Colima: Hyw Colima- 6 July 2008 venulosa Minatitlan 7 July 2008 Smilisca baudinii Colima: Hyw 98 Minatitlan -Manzanillo Rhabditida Diaphanocephalidae* Kalicephalus sp. 2 (2) Leptodeira sp. Colima: Comala 27 June 2009 Imantodes sp. Colima: Ixtlahuacan 24 June 2009 Spirurida Heterakidae Strongyluris sp. 1 (1) Trimorphodon Colima: Hwy 98 Minatitlan 8 July 2008 biscutatus -Manzanillo Spirurida Pharyngodonidae Ozolaimus sp. 5 (0) Ctenosaura sp. Colima: Hyw 54 Ixtlahuacan 8 July 2008 Spirurida Pharyngodonidae Parapharyngodon sp. 4 (4) Phrynohyas Colima: Hyw 98 Minatitlan 8 July 2008 venulosa -Manzanillo Spirurida Pharyngodonidae gen sp. 1 15 (10) Sceloporus sp. Jalisco: ND 25 July 2009 Spirurida Pharyngodonidae gen sp. 2 2 (2) Sceloporus formosus Veracruz: Hyw Xico 28 July 2004 Viejo- Matlalapa Spirurida Cosmocercidae Aplectana sp. 18 (17) Rana pustulosa Nayarit: S of Hyw Barranca 24 June 2009 Bufo sp. del Oro: Barranqueno~ bridge Leptodeira sp. Nayarit: Hyw Uzeta-La Gloria Colima: Comala Spirurida Onchocercidae Foleyellides sp. 11 (11) Rana pustulosa Nayarit: S of Hyw Barranca 24 June 2009 Rana psilonota del Oro: Barranqueno~ bridge 30 June 2010 © Jalisco: Zapopan: Barranca 2013 Blackwell Publishing Ltd del rıo Santiago Spirurida Physalopteridae Physaloptera sp. 7 (7) Trimorphodon Michoacan: Hwy 200 5 July 2008 biscutatus between La placita and Maruata Spirurida Physalopteridae gen sp. 1 5 (5) Sceloporus sp. Jalisco: ND 25 June 2009 Spirurida Physalopteridae gen sp. 2 1 (1) Imantodes sp. Colima: Hyw Comala 25 June 2009 -Minatitlan Spirurida Physalopteridae Turgida sp. 1 (1) Didelphis virginiana Jalisco: Zapopan: Barranca 30 June 2010 del rıo Santiago PRIMERS FOR NEMATODE DNA BARCODING 3 requires the amplification of multiple regions to be effec- Materials and methods tive (De Ley et al. 2005; Subbotin et al. 2008). Similar studies using ITS revealed that a lack of phylum-wide Specimen collection primers combined with difficulties in aligning the extre- mely variable ITS sequences precluded its use as a Ninety-five adult nematodes collected in Mexico from universal nematode identification marker amenable to various reptilian, amphibian and mammalian hosts were high-throughput platforms (Floyd et al. 2002; De Ley analysed (Table 1). Each specimen was collected in et al. 2005). duplicate (i.e. from the same habitat within the same The mitochondrial gene cyctochrome c oxidase sub- host), with one stored in 95% ethanol for DNA extraction unit I (COI) has also been explored as a potential marker and the other cleared on a glass slide with undiluted on which to base a nematode phylogenetic classification glycerine to enable identification to family, genus or spe- system (Floyd et al. 2002; Elsasser et al. 2009). In addition cies level using morphological characteristics (Table 1). to being a mitochondrial gene, COI is translated into an evolutionarily conserved protein and thus has some Primer design advantages over SSU, LSU and ITS. However, COI is not immune to the inherent problems associated with nema- Cytochrome c oxidase subunit I (COI) sequences were tode barcoding. While the 5′ region of COI has been obtained from 56 mitochondrial genome sequences from shown to separate nematodes into proper species (Dery- nematodes in GenBank (Table 2) and aligned using cke et al. 2010), a phylum-wide primer set has yet to be online EBI CLUSTALW2 software (Larkin et al. 2007). A developed (De Ley et al. 2005). In this study, we report lepidopteran COI sequence was included in the align- the development of a primer cocktail which enables ment as a reference for locating the standard primer the recovery of COI barcodes from a broad range of binding sites (Folmer et al. 1994) for COI barcoding nematode parasites of vertebrates in a high-throughput (Hebert et al. 2003a,b). The forward and reverse primer manner and delivers species-level resolution. binding sites were excised from the 56 sequences and Table 2 Nematode COI sequences used to design cocktail primers GenBank Accession Species GenBank Accession Species NC_008231 Agamermis sp. BH-2006 AJ556134 Necator americanus FJ483518 Ancylostoma caninum NC_003416 Necator americanus NC_003415 Ancylostoma duodenale GQ888716 Oesophagostomum dentatum GQ398121 Angiostrongylus cantonensis FM161883 Oesophagostomum quadrispinulatum GQ398122 Angiostrongylus costaricensis NC_001861 Onchocerca volvulus NC_007934 Anisakis simplex FN313571 Radopholus similis NC_001327 Ascaris suum NC_008640 Romanomermis culicivorax